Programmed cell death is an integral component ofC. elegansdevelopment. Genetic studies inC. eleganshave led to the identification of more than two dozen genes that are important for the specification of which cells should
live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular and biochemical
studies have revealed the underlying conserved mechanisms that control these three phases of programmed cell death. In particular,
an interplay of transcriptional regulatory cascades and networks involving CES-1, CES-2, HLH-1/HLH-2, TRA-1, and other transcriptional regulators is crucial in activating the expression of the key death-inducing geneegl-1in cells destined to die. A protein interaction cascade involving EGL-1, CED-9, CED-4 and CED-3 results in the activation of the key cell death protease CED-3. The activation of CED-3 initiates the cell disassembly process and nuclear DNA fragmentation, which is mediated by the release of apoptogenic mitochondrial
factors (CPS-6 and WAH-1) and which involves multiple endo- and exo-nucleases such as NUC-1 and seven CRN nucleases. The recognition and removal of the dying cell is mediated by two partially redundant signaling pathways
involving CED-1, CED-6 and CED-7 in one pathway and CED-2, CED-5, CED-10, CED-12 and PSR-1 in the other pathway. Further studies of programmed cell death inC. eleganswill continue to advance our understanding of how programmed cell death is regulated, activated, and executed in multicellular
organisms.

1. Introduction

Genetic studies of programmed cell death, or apoptosis, in C. elegans led to the identification of key players involved in this important physiological process from C. elegans to humans (Adams, 2003; Danial and Korsmeyer, 2004; Horvitz, 2003). These pioneering studies were made possible by the biology of C. elegans: 1. Unlike in many other animals, programmed cell death is not essential for C. elegans viability, at least under laboratory conditions (Ellis and Horvitz, 1986); 2. cells undergoing programmed cell death in C. elegans change their morphology and refractivity and can be observed in living animals using Differential Interference Contrast microscopy
(DIC), also referred to as Nomarski optics (Figure 1; Robertson and Thomson, 1982); 3. programmed cell death that occurs during C. elegans development is determined by the essentially invariant somatic cell lineage of C. elegans; therefore, it is not only known which cells undergo programmed cell death but also when and where they die (Sulston and Horvitz, 1977; Sulston et al., 1983). These unique features made it possible to genetically dissect the process of programmed cell death in C. elegans at single cell resolution. The resulting ground-breaking work was recognized with the Nobel Prize for Medicine in 2002, which
was awarded to Sydney Brenner, John E. Sulston, and H. Robert Horvitz for their leading roles in deciphering the C. elegans cell lineage and in defining the genetic pathway of programmed cell death (Brenner, 2003; Horvitz, 2003; Sulston, 2003).

Figure 1. Nomarski image of an embryo with apoptotic cells. Three cells indicated by arrows underwent programmed cell death in a bean/comma stage
embryo and exhibit a refractile, raised-button-like appearance. The bar represents 5 μm.

Programmed cell death occurs during two stages of C. elegans life and in two different types of tissues: during embryonic and post-embryonic development of the soma (referred to as "developmental
cell death"; Sulston and Horvitz, 1977; Sulston et al., 1983) and in the gonad of adult hermaphrodites (referred to as "germ cell death"; Gumienny et al., 1999; Sulston, 1988; White, 1988). Programmed cell death proceeds in three genetically distinguishable phases: during the "specification phase", a cell is
instructed to undergo programmed cell death; in the "killing phase", the apoptotic program is activated in the cell instructed
to die; during the "execution phase", cells are dismantled and subsequently engulfed by neighboring cells (Horvitz, 1999; Figure 2). Mutations that lead to a partial block in this final phase, such as mutations in the genes ced-1 and ced-2 (Hedgecock et al., 1983), result in the accumulation of dead cells (referred to as "cell corpses"). Mutations in ced-1 and ced-2 were the first mutations to be identified as affecting programmed cell death and they were instrumental in the subsequent
identification of genes involved in apical phases of programmed cell death. In the following, we will review our current understanding
of the genes involved in the specification, killing, and execution phases in the case of developmental cell death. Germ cell
death is discussed elsewhere in this book (see Germline survival and cell death).

Figure 2. Genetic pathway of programmed cell death in C. elegans. Three phases of programmed cell death, specification, killing, and execution, are
indicated. In the "specification" phase, genes involved in regulating the death fates of specific cells (HSNs and NSM sister cells) are shown. In the "execution" phase, two partially redundant pathways mediate the engulfment of cell corpses and the fragmentation of chromosomal DNA.
nuc-1 and crn-6 may be involved in the degradation of DNA debris from apoptotic cells.

2. Killing phase

2.1. The core machinery involved in the activation of the apoptotic program

In addition to egl-1, ced-3, ced-4 and ced-9, several other genes have been implicated in the activation of the apoptotic program during C. elegans development, including the dad-1 gene (dad, defender against apopototic death; Sugimoto et al., 1995), which encodes a protein similar to the mammalian DAD1 protein (Nakashima et al., 1993), and the icd-1 gene (icd-1, inhibitor of cell death; Bloss et al., 2003; Sugimoto et al., 1995), which encodes a protein similar to the beta-subunit of the nascent polypeptide-associated complex (betaNAC). How dad-1 and icd-1 might interact with the core killing machinery is currently unclear.

Figure 3. Biochemical model for the activation of programmed cell death. (A) In living cells, CED-4 is tethered to the surface of mitochondria through binding
to CED-9. (B) In cells that are doomed to die, the death initiator EGL-1 binds to CED-9,
causes a major CED-9 conformational change, and triggers the disassociation of CED-4
from CED-9. (C) Released CED-4 proteins translocate to perinuclear membranes and undergo
oligomerization, which brings two CED-3 proenzymes to close proximity. (D) CED-3
proenzymes undergo autoproteolytic activation.

How does a cell know whether to live or die? In higher organisms, cells are generally instructed to die by neighboring cells
or by extracellular cues. In contrast, most of the 131 cell deaths observed during C. elegans development appear to occur in a cell-autonomous manner; i.e. cells appear to "know" at the time of their birth whether their
"fate" is to live or die (Sulston and White, 1980; Evidence for cell non-autonomous induction of cell death exists however in the germ line of C. elegans hermaphrodites; see Germline survival and cell death). The observations that cell fate altering mutations, such as loss-of-function mutations of unc-86 (unc, uncoordinated) or pag-3 (pag, pattern of reporter gene expression abnormal), can affect the essentially invariant pattern of programmed cell death suggest that programmed cell
death can be regarded as a cell fate (Cameron et al., 2002; Chalfie et al., 1981; Finney et al., 1988; Sulston and Horvitz, 1981). The current model for cell death specification is that in the 959 cells destined to survive, EGL-1 activity is low or absent and that in the 131 cells destined to die, EGL-1 activity is high. High EGL-1 activity inhibits CED-9 activity, resulting in the activation of CED-4 and CED-3 and the commitment of a cell to the cell death fate (Horvitz, 2003).

At least some of the regulators of egl-1 expression have additional functions during development. For example, TRA-1 is the terminal, global regulator of somatic sexual fate required for female development (Hodgkin, 1987; Zarkower and Hodgkin, 1992) and HLH-2 is essential for viability (Krause et al., 1997). Some of the cascades or networks that control egl-1 expression during development appear to be conserved: CES-1-like members of the Snail family of DNA-binding proteins, such
as Snail and SLUG, can confer resistance to programmed cell death in mammals and in chick (Inoue et al., 2002; Inukai et al., 1999; Perez-Losada et al., 2003; Vega et al., 2004), and the CES-2-like proto-oncogene HLF (HLF, hepatic leukemia factor) has been implicated in the regulation of SLUG in mammals
(Inaba et al., 1996; Inukai et al., 1999). However, egl-1 might not be the only gene that needs to be regulated at the transcriptional level for proper cell death specification. The
proteins EOR-1 and EOR-2 (EOR, enhancer of Raf), which have been proposed to act as regulators of transcription (Howard and Sundaram, 2002), are required for the death of the HSNs in males, however, they are not required for the activation of egl-1 transcription in these cells (Hoeppner et al., 2004).

4. Execution phase

Once the apoptotic program is activated, it initiates the cell disassembly process, which includes nuclear DNA fragmentation,
cytoplasm shrinkage, and exposure of "eat-me" signal(s) on the cell surface to induce phagocytosis by neighboring cells (Steller, 1995).

4.1. Nuclear DNA fragmentation

The fragmentation of chromosomal DNA is a hallmark of apoptosis and may facilitate apoptosis by terminating DNA replication
and gene transcription (Arends et al., 1990). DNA fragmentation during C. elegans apoptosis has been studied with the aid of various DNA-staining techniques, including DAPI or Feulgen staining (Sulston, 1976) or TUNEL staining (Gavrieli et al., 1992; Wu et al., 2000).

So far ten genes have been identified to be involved in nuclear DNA degradation during apoptosis (Parrish et al., 2001; Parrish and Xue, 2003; Sulston, 1976; Wang et al., 2002; Wu et al., 2000). These include nuc-1 (nuc, nuclease defective), cps-6 (cps, CED-3protease suppressors), wah-1 (wah, worm AIF homologue), crn-1 to crn-6 (crn, cell death related nucleases), and cyp-13 (cyp, cyclophilins). Loss or reduction of activity in any of these genes results in the accumulation of TUNEL-positive cells in C. elegans embryos, suggesting that these genes are important for resolving TUNEL-reactive DNA breaks generated during apoptosis (Parrish et al., 2001; Parrish and Xue, 2003; Sulston, 1976; Wang et al., 2002; Wu et al., 2000). In addition, reduction of activity in most of these genes (with the exception of nuc-1 and crn-6) causes delayed appearance of embryonic cell corpses during development and reduced cell deaths in sensitized genetic backgrounds,
suggesting that nuclear DNA degradation is important for normal progression of the apoptotic process and can even promote
cell killing. Genetic and phenotypic analyses indicate that these genes act in multiple pathways and at different stages to
promote DNA degradation and apoptosis, with cps-6, wah-1, crn-1, crn-4, crn-5 and cpy-13 acting in one pathway and crn-2 and crn-3 in the other (Parrish and Xue, 2003; Wang et al., 2002). Defects in both DNA degradation pathways not only cause a more severe defect in nuclear DNA degradation but also a synthetic
defect in cell corpse engulfment, suggesting that the DNA degradation process may affect cell corpse removal (Parrish and Xue, 2003). In addition to its cell death function, nuc-1 is involved in the degradation of DNA derived from ingested bacteria in the intestinal lumens (Sulston, 1976; Wu et al., 2000).

Both cps-6 and wah-1 encode mitochondrial proteins, which are similar to human mitochondrial endonuclease G (EndoG) and apoptosis-inducing factor
(AIF), respectively (Parrish et al., 2001; Wang et al., 2002). Ectopic egl-1 expression induces WAH-1 translocation from mitochondria to nuclei in a CED-3 dependent manner, suggesting that the role of mitochondria in regulating apoptosis is conserved. The WAH-1 protein can physically associate with CPS-6 and enhance the endonuclease activity of CPS-6 (Wang et al., 2002). Furthermore, CPS-6, CRN-1, CRN-4, CRN-5 and CYP-13, which are either endonucleases or exonucleases, appear to interact and cooperate with one another, possibly in a large DNA
degradation complex named degradeosome (Parrish and Xue, 2003), to promote stepwise DNA fragmentation, starting from generating DNA nicks, gaps, to double-stranded DNA breaks (Parrish et al., 2003). Both nuc-1 and crn-6 encode type II acidic DNases and do not seem to affect either the activation or progression of cell death or the engulfment
of cell corpses (Hedgecock et al., 1983; Parrish et al., 2001; Parrish and Xue, 2003; Wu et al., 2000). These two nucleases may act at later stages of apoptotic DNA degradation, possibly in the lysosomal compartments of the
engulfing cell to promote degradation of engulfed apoptotic cells.

Figure 4. Molecular model for the cell corpse engulfment process. The engulfment process is mediated by two partially redundant pathways. In the
CED-1/CED-6/CED-7 pathway, CED-1 and CED-7 act on the surface of the engulfing cell to
mediate recognition of an unknown engulfment signal(s) on the surface of the dying cell
(green diamonds) and to transduce the signal through CED-6 to activate the phagocytic
machinery of the engulfing cell. CED-7 also acts in dying cells. In the CED-2, CED-5,
CED-10 and CED-12 pathway, PSR-1 may act in the engulfing cell to mediate the
recognition of PS (red circles) externalized by the dying cell and to transduce the
signal through the CED-2/CED-5/CED-12 ternary complex to activate CED-10. There are
likely other engulfment receptors that act in the CED-2, CED-5, CED-10 and CED-12
pathway.

Phagocytosis not only removes cell corpses generated by programmed cell death, but also may actively promote apoptosis (Hoeppner et al., 2001; Reddien et al., 2001). Engulfment genes appear to act in engulfing cells to promote apoptosis (Reddien et al., 2001). Furthermore, these two engulfment pathways are also important for the removal of necrotic cell corpses, suggesting that
similar mechanisms are used to recognize and remove apoptotic and necrotic corpses (Chung et al., 2000). Several other genes have also been implicated in cell death execution, including the ced-8 gene which appears to control the timing of cell death and encodes a protein similar to human XK, a putative membrane transport
protein (Stanfield and Horvitz, 2000).

5. Conclusions

The genetic and molecular characterization of genes, which, when mutated or inactivated by RNAi, affect developmental cell
death in C. elegans, has revealed some of the molecular mechanisms involved in the specification, killing or execution phase of programmed cell
death. However, much remains to be learnt about programmed cell death. For example, while we know that the transcriptional
activation of the egl-1 gene specifies whether a cell will live or die, very little is known about what regulates egl-1 expression. Furthermore, while we know that the CED-3 caspase is essential for cell killing and required for DNA fragmentation and engulfment, it is currently not known how CED-3 is activated and what CED-3 substrates are. Finally, while more is known about the molecular components that act in engulfing cells to mediate cell corpse
engulfment, little is known about what acts in the dying cell to trigger the phagocytic event and how engulfing cells promote
killing in a cell non-autonomous manner. By answering these and other remaining questions, studies of developmental cell death
in C. elegans will continue to contribute in a major way to our current knowledge of programmed cell death.

6. Acknowledgements

We would like to thank Yi-chun Wu for contributing some of the figures and for writing a portion of this chapter. Research
in Ding Xue's laboratory is supported by a Searle Scholar award, a Burroughs Wellcome Fund Career Award, and grants from NIH
and Department of Defense. Research in Barbara Conradt's laboratory is supported by the Howard Hughes Medical Institute (HHMI
award to Dartmouth Medical School under the Biomedical Research Support Program for Medical Schools) and NIH.

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